The proposed studies make use of viruses collected from injection drug users (IDUs) to address two high priority research objectives for HIV vaccine research. These are: 1) Defining the specificities of antibodies that neutralize diverse primary isolates, and 2) Determining why broadly neutralizing antibodies are uncommon and how they can be elicited. Broadly neutralizing antibodies (bNAbs) able to neutralize a wide range of clinical isolates of HIV are found in sera from ~10-30% of HIV-1 infected individuals (HIV+ sera). However, despite years of investigation, the epitopes recognized by bNAbs remain poorly defined. In this proposal, we apply a new method, swarm analysis, recently developed in our lab, to a unique set of clinical specimens obtained from injection drug users (IDUs) in Bangkok, Thailand. The results from these studies should allow us for the first time to map the epitopes recognized by bNAbs in HIV+ sera, and to identify mutations that affect sensitivity and resistance to neutralization. We have demonstrated the feasibility of this approach in preliminary studies, and have used it to identify a new mutation in gp41 that confers sensitivity to neutralization by bNAbs. This information will be useful for the development of novel antigens designed to elicit bNAbs similar to those found in HIV+ sera. The special circumstance of HIV transmission in IDUs, where groups of genetically diverse individuals are infected with the same virus through needle sharing (IDU transmission cohorts), provides a unique opportunity to maximize the information collected from the swarm analysis and better define the performance requirements for a successful HIV vaccine. The fact that two clades of HIV: E (A/E recombinant) and B' co-circulate in the Thai cohort allows us to focus on bNAbs with broad, cross-clade neutralizing activity. Previous efforts to identify epitopes recognized by bNAbs relied on production of monoclonal antibodies. However, after more than 20 years of effort, only a few monoclonal antibodies with potent broadly neutralizing activity have been identified. Moreover, the physiologic relevance of these monoclonal antibodies remains uncertain, since it has been difficult to demonstrate that HIV-infected humans produce antibodies with similar specificities. Our new method of epitope mapping makes use of the swarm of closely related virus variants that occur within each HIV-infected individual as a source of naturally occurring mutants that can be used to map epitopes. This method alleviates concerns regarding physiologic relevance by focusing on antibodies and virus mutants that evolve during the normal course of HIV infection. The proposed studies provide a unique, stepwise back to the drawing board approach to HIV vaccine development.